Therapeutic agent for motor neuron disease

ABSTRACT

An object of the present invention is to provide an agent effective for the treatment and/or prevention of motor neuron disease such as amyotrophic lateral sclerosis (ALS). The present invention provides a therapeutic and/or preventive agent for motor neuron disease comprising the following oligopeptide shown in any of (a) to (c) or a pharmaceutically acceptable salt thereof as an active ingredient: (a) an oligopeptide consisting of the amino acid sequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); (b) an oligopeptide consisting of an amino acid sequence having a deletion, substitution, insertion, or addition of one or several amino acids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and having an activity that inhibits neuronal cell death caused by a mutant superoxide dismutase-1 gene; and (c) a modified oligopeptide from the oligopeptide (a) or (b).

TECHNICAL FIELD

The present invention relates to a therapeutic and/or preventive agentfor motor neuron disease, particularly amyotrophic lateral sclerosis(ALS).

BACKGROUND ART

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease thattypically affects people of middle or advanced ages and selectivelyattacks motor nerves in the cerebrum, brain stem, and spinal cord(Cleveland D W and Rothstein J D, 2001, Nat Rev Neurosci 2: 806-819; andHand C K and Rouleau G A, 2002, Muscle Nerve 25: 135-159). ALS causesmuscular atrophy and muscular weakness in voluntary muscles in the wholebody except for extraocular muscle, and eventually respiratory failure.Most patients die in 3 to 5 years from the onset.

Riluzole is the sole drug previously approved for ALS in US and Japan.Riluzole was originally developed as an anticonvulsant inhibitingglutamate release and has been reported in several clinical trials toexhibit only slight efficacy for the survival of ALS patients (Rowland LP and Shneider N A, 2001, N Engl J Med, 344, 1688-1700; and Turner M Rand Parton M J, 2001, Semin Neurol 21: 167-175). Besides riluzole,mutiple factors including ciliary neurotrophic factor (CNTF) andinsulin-like growth factor I (IGF-I) were tested in clinical trials and,however, fell short of success (Miller R G et al., 1996, Ann Neurol 39:256-260). Thus, there are no therapeutic agents effective for ALS underpresent circumstances.

Approximately 10% of ALS cases are familial (FALS) and most FALS casesare inherited autosomal-dominantly. In 1993, Rosen et al identified forthe first time the superoxide dismutase-1 (SOD1) gene located on thechromosome 21 as a causative gene by analyzing pedigree with autosomalinheritance (Rosen D R, et al., 1993, Nature 362: 59-62). Approximately20% of FALS cases are caused by mutations in the SOD1 gene, and most ofthese mutations are missense point mutations. 100 or more mutations inSOD1 caused FALS (Cleveland D W and Rothstein L D, supra). Severalgroups have reported that overexpression of FALS-associated SOD1 mutantgene induces neuronal cell death in vitro (e.g., Rabizadeh S, et al.,1995, Proc Natl Acad Sci USA 92: 3024-3028; Durham H D et al., 1997, JNeropathol Exp Neurol 56: 523-530; and Ghadge G D et al., 1997 J Nerosci17: 8756-8766). Besides this, the activation of caspase-3 has beenobserved in the spinal cords in ALS patients. Accordingly, theinhibition of neuronal cell death is an important strategy to developtherapeutic agents for ALS.

In addition to CNTF and IGF-I described above, Bcl2, a non-specificcaspase inhibitor zVAD-fmk (Kostic V, et al., 1997, Science 277:559-562; Azzouz M, et al., 2000, Hum Mol Genet. 9: 803-811; and Li M, etal., 2000, Science 288: 335-339), and alsin, the newly found product ofALS2 gene causative of recessive inherited FALS (Kanekura K, et al,2004, J Biol Chem 279: 19247-19256), have been reported so far as thoseexhibiting inhibitory (antagonistic) action on neuronal cell deathcaused by the overexpression of SOD1 mutants.

On the other hand, ADNF or ADNF9 (activity-dependent neurotrophicfactor), which is a short peptide consisting of nine amino acid residues(Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala), was originally purified by Gozeset al from the culture medium of astrocytes stimulated with VIP(Brenneman D E and Gozes I, 1996, J Clin Invest 97: 2299-2307; BrennemanD E, et al., 1998, J Pharmacol Exp Ther 285: 619-627; and Blondel O, etal., 2000, J Neurosci 20: 8012-8020). ADNF was shown to protect neuronsfrom death caused by some neurological disorders including amyloid β(Brenneman D E, et al., 1998, J Pharmacol Exp Ther 285: 619-627; andGlazner G W, et al., 2000, J Neurochem 73: 2341-2347). ADNF is aneuroprotective factor unique in that it has activity at its lowerconcentrations of femtomolar to picomolar levels, and loses itsprotective effect at higher concentrations above the nanomolar order.This unique but unfavorable property of ADNF have prevented it frombeing developed as an anti-Alzheimer's disease (AD) drug.

The object of the present invention is to provide an agent that inhibitsneuronal cell death causing ALS and is effective for the treatment ofamyotrophic lateral sclerosis.

DISCLOSURE OF THE INVENTION

We have now conducted diligent studies to attaint the object and haveconsequently completed the present invention by finding out that anactivity-dependent neurotrophic factor (hereinafter, referred to as“ADNF”) significantly inhibits (antagonizes) neuronal cell death inducedby mutant superoxide dismutase-1 (SOD1) genes and has the effect ofimproving the motor function of ALS model mice and delaying ALS onset.

Namely, the present invention encompasses the following inventions.

(1) A therapeutic and/or preventive agent for motor neuron diseasecomprising the following oligopeptide shown in any of (a) to (c) or apharmaceutically acceptable salt thereof as an active ingredient:

(a) an oligopeptide consisting of the amino acid sequence represented bySer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1);

(b) an oligopeptide consisting of an amino acid sequence having adeletion, substitution, insertion, or addition of one or several aminoacids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and havingan activity that inhibits neuronal cell death caused by a mutantsuperoxide dismutase-1 gene; and

(c) a modified oligopeptide from the oligopeptide (a) or (b).

(2) A therapeutic and/or preventive agent for motor neuron diseasecomprising DNA encoding the following oligopeptide (a) or (b) as anactive ingredient:

(a) an oligopeptide consisting of the amino acid sequence represented bySer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); and

(b) an oligopeptide consisting of an amino acid sequence having a adeletion, substitution, insertion, or addition of one or several aminoacids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and havingan activity that inhibits neuronal cell death caused by a mutantsuperoxide dismutase-1 gene.

(3) The agent according to (1) or (2), wherein the motor neuron diseaseis amyotrophic lateral sclerosis (ALS).(4) The agent according to (1) or (2), wherein the agent has the effectof delaying the onset of the motor neuron disease and improving themotor function of a patient with the motor neuron disease.(5) A fusion peptide of the following oligopeptide (a) or (b) withanother peptide:

(a) an oligopeptide consisting of the amino acid sequence represented bySer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); and

(b) an oligopeptide consisting of an amino acid sequence having adeletion, substitution, insertion, or addition of one or several aminoacids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1) with the andhaving an activity that inhibits neuronal cell death caused by a mutantsuperoxide dismutase-1 gene.

(6) The fusion peptide according to (5), wherein the another peptide isa signal peptide for extracellular secretion and/or tag peptide forpurification/detection.(7) DNA encoding the following oligopeptide (a) or (b) or a fusionpeptide according to (5):

(a) an oligopeptide consisting of the amino acid sequence represented bySer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); or

(b) an oligopeptide consisting of an amino acid sequence having adeletion, substitution, insertion, or addition of one or several aminoacids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and havingan activity that inhibits neuronal cell death caused by a mutantsuperoxide dismutase-1 gene.

(8) A recombinant vector comprising DNA according to (7).(9) A host cell transformed with DNA according to (8).(10) A method for producing the following oligopeptide (a) or (b),characterized by culturing a host cell according to (9) in a medium andcollecting an oligopeptide expressed from the obtained culture:

(a) an oligopeptide consisting of the amino acid sequence represented bySer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); or

(b) an oligopeptide consisting of an amino acid sequence having adeletion, substitution, insertion, or addition of one or several aminoacids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and havingan activity that inhibits neuronal cell death caused by a mutantsuperoxide dismutase-1 gene.

The present invention also encompasses a use of the oligopeptide shownin any of (a) to (c) or a pharmaceutically acceptable salt thereof forthe production of said agent; and a method for treating a motor neurondisease comprising the step of administering an effective amount of theoligopeptide shown in any of (a) to (c) or a pharmaceutically acceptablesalt thereof to a mammal including human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) shows the cell mortality of a neuronal cell line (F11 cell)transformed with a control vector (pEF-BOS vector) or wild-type SOD1gene (wt-SOD1). FIG. 1(B) shows the cell mortality of a neuronal cellline (F11 cell) transformed with a control vector (pEF-BOS) or SOD1mutant (G85R-SOD1) gene. FIG. 1(C) shows the cell mortality of aneuronal cell line (F11 cell) transformed with a control vector(pEF-BOS), wild-type SOD1 gene (wt-SOD1), or SOD1 mutant (A4T-SOD1,G85R-SOD1, or G93R-SOD1) gene, in the presence or absence of ADNF orHNG;

FIG. 2A shows the dose-dependent inhibitory effect of ADNF on cell deathinduced by a neuronal cell line (F11 cell) transformed with a SOD1mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) gene. The lower panel showsthe expression level of SOD1 proteins detected by immunoblot;

FIG. 2B shows the dose-dependent inhibitory effect of ADNF on cell deathinduced by a neuronal cell line (NSC34 cell) transformed with a SOD1mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) gene. The lower panel showsthe expression level of SOD1 proteins detected by immunoblot;

FIG. 3A shows the effect of wortmannin (W), genistein (G), PD98059 (PD),SB203580 (SB), AG490 (AG), KN93 (KN), or HA1004 (HA) on cell deathinduced by a neuronal cell line (F11 cell) transformed with a controlvector (pEF-BOS) or with a SOD1 mutant (A4T-SOD1) gene, in the presenceor absence of 100 nM ADNF. The lower panel shows the expression level ofSOD1 proteins detected by immunoblot;

FIG. 3B shows the effect of wortmannin (W), genistein (G), PD98059 (PD),SB203580 (SB), AG490 (AG), KN93 (KN), or HA1004 (HA) on cell deathinduced by a neuronal cell line (F11 cell) transformed with a controlvector (pEF-BOS) or with a SOD1 mutant (G85R-SOD1) gene, in the presenceor absence of 100 nM ADNF. The lower panel shows the expression level ofSOD1 proteins detected by immunoblot;

FIG. 3C shows the effect of wortmannin (W), genistein (G), PD98059 (PD),SB203580 (SB), AG490 (AG), KN93 (KN), or HA1004 (HA) on cell deathinduced by a neuronal cell line (F11 cell) transformed with a controlvector (pEF-BOS) or with a SOD1 mutant (G93R-SOD1) gene, in the presenceor absence of 100 nM ADNF. The lower panel shows the expression level ofSOD1 proteins detected by immunoblot;

FIG. 4A shows the effect of KN93 or KN92 on cell death induced by aneuronal cell line (F11 cell) transformed with a control vector(pEF-BOS) or with a SOD1 mutant (A4T-SOD1) gene, in the presence orabsence of 100 μM ADNF. The lower panel shows the expression level ofSOD1 protein detected by immunoblot;

FIG. 4B shows the effect of KN93 or KN92 on cell death induced by aneuronal cell line (F11 cell) transformed with a control vector(pEF-BOS) or with a SOD1 mutant (G85R-SOD1) gene in the presence orabsence of 100 fM ADNF. The lower panel shows the expression level ofSOD1 protein detected by immunoblot;

FIG. 4C shows the effect of KN93 or KN92 on cell death induced by aneuronal cell line (F11 cells) transfected with a control vector(pEF-BOS) or with a SOD1 mutant (G93R-SOD1) gene in the presence orabsence of 100 fM ADNF. The lower panel shows the expression level ofSOD1 proteins detected by immunoblot;

FIG. 4D shows the cell death of F11 cells, which were cotransformed witha SOD1 mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) gene and with akinase-inactive CaMKII or CaMKIV cDNA, in the presence or absence of 100nM ADNF;

FIG. 5A shows the effect of an IPAL peptide (IPALDSLKPANEDQKIGIEI) oncell death induced by a neuronal cell line (F11 cell) transformed with acontrol vector (pEF-BOS vector) or with a SOD1 mutant (A4T-SOD1,G85R-SOD1, or G93R-SOD1) gene in the presence or absence of 100 fM ADNF;

FIG. 5B shows the cell mortality of a neuronal cell line (F11 cell)transformed with a control vector (pEF-BOS) or with a SOD1 mutant(G93R-SOD1) gene in the presence of varying concentrations of ADNF(SALLRSIPA) or ADNF8 (ALLRSIPA); and

FIG. 6 shows the effect of icv injection of ADNF on the motor functionof G93A-SOD1 transgenic mice.

Hereinafter, the present invention will be described in detail. Thepresent application claims the priority of U.S. Provisional ApplicationNo. 60/560, 254 filed on Apr. 8, 2004 and encompasses contents asdescribed in the specification and/or drawings of the priorityapplication.

1. Agent of the Invention

An oligopeptide, as an active ingredient, in the agent of the presentinvention is an oligopeptide consisting of the amino acid sequencerepresented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1).

The oligopeptide also encompasses a mutant oligopeptide having adeletion, substitution, insertion, or addition of one or several aminoacids in said amino acid sequence as long as the mutant oligopeptide hasan activity that inhibits neuronal cell death caused by a mutantsuperoxide dismutase-1 (hereinafter, referred to as “SOD1 mutant) gene.The range of the “one or several” is not particularly limited and means,for example, one to five, preferably one to three, more preferably oneor two amino acids.

The substation by another amino acid can include the substitutionbetween hydrophobic amino acids (Ala, Ile, Leu, Met, Phe, Pro, Trp, Thr,and Val), between hydrophilic amino acids (Arg, Asp, Asn, Cys, Glu, Gln,Gly, H is, Lys, Ser, and Thr), between amino acids with aliphatic sidechains (Gly, Ala, Val, Leu, Ile, and Pro), or between amino acids withhydroxy group-containing side chains (Ser, Thr, and Tyr). However, thesesubstitutions are merely illustrated for preferable examples, and otheramino acid substitutions may be performed as long as the resultingmutant oligopeptide maintains an activity that inhibits neuronal celldeath caused by a SOD1 mutant gene.

The SOD1 mutant gene refers to a gene encoding SOD1 comprising asubstitution of Ala at position 4 (corresponding to position 5 in SEQ IDNO: 5) by Thr, a substitution of Gly at position 85 (corresponding toposition 86 in SEQ ID NO: 5) by Arg, or a substitution of Gly atposition 93 (corresponding to position 94 in SEQ ID NO: 5) by Arg,provided that the position of Ala following the initiation codon (Met)is numbered 1, in the wild-type human SOD1 amino acid sequence (SEQ IDNO: 5).

The oligopeptide and an altered oligopeptide thereof may be modifiedchemically or biologically. Examples of the modification can include,but are not limited to, functional group introduction such asalkylation, esterification, halogenation, or amination, functional groupconversion such as oxidation, reduction, addition, or elimination, theintroduction of sugar compounds (monosaccharide, disaccharide,oligosaccharide, or polysaccharide) or lipid compounds, phosphorylation,and biotinylation.

Those skilled in that art can confirm by test methods as describedspecifically and in detail in Examples later or by appropriately alteredor modified versions of the test methods that the altered oligopeptideor modified oligopeptide has an activity that inhibits cell death causedby a SOD1 mutant gene, which activity is analogous to the inhibitoryactivity of the oligopeptide comprising the amino acid sequencerepresented by SEQ ID NO: 1.

The oligopeptides are of various types (including altered or modifiedoligopeptides), and they may be in a free form or in an acid- orbase-addition salt. Examples of the acid-addition salt can include saltsof mineral acids such as hydrochloride, sulfate, nitrate, and phosphate;and salts of organic acids such as citrate, oxalate, maleate, andtartrate. Examples of the base-addition salt can include metal saltssuch as sodium salts, potassium salts, calcium salts, and magnesiumsalts; ammonium salts; organic ammonium salts such as methylammoniumsalts and triethylammonium salts.

Furthermore, these oligopeptides or salts thereof sometimes exist ashydrates or solvates.

The oligopeptides can be synthesized by a routine peptide synthesismethod known in the art. Specifically, they can be synthesized by avariety of methods such as azide method, acid chloride method, acidanhydride method, mixed anhydride method, DCC method, activated estermethod (e.g., P-nitrophenyl ester, N-hydroxysuccinimide ester, andcyanomethyl ester methods), methods using Woodward's reagent K,carboimidazole method, oxidation-reduction method, and DCC-additive(HONB, HOBt, or HOSU) method according to the descriptions of, forexample, “The Peptides” Vol. 1 (1966) [Schroder and Lubke, AcademicPress, New York, U.S.A.] or “Peptide Synthesis” [Izumiya et al., MaruzenCo., Ltd., (1975)]. These methods can be applied to both solid-phase andliquid-phase syntheses.

In the solid-phase method, a variety of commercially available peptidesynthesizers can be utilized. The synthesis can be performed moreefficiently by protecting and deprotecting functional groups, ifnecessary. For example, Protective Groups in Organic Synthesis (T. W.Greene, John Wiley & Sons Inc., 1981) can be referenced for proceduresfor introducing and eliminating protecting groups.

The obtained oligopeptide can be desalted and purified according to atypical method. Examples thereof include ion-exchange chromatographysuch as DEAE-cellulose, partition chromatography such as Sephadex LH-20and Sephadex G-25, normal phase chromatography such as silica gel,reverse phase chromatography such as ODS-silica gel, and highperformance liquid chromatography.

The oligopeptide inhibits neuronal cell death caused by a SOD1 mutantgene and has neuroprotective action. The neuroprotective action of theoligopeptide is attributed to a mechanism (novel mechanism) differentfrom that of antagonistic action on neuronal cell death caused bypreviously reported amyloid β toxicity because the action was notinhibited by IPAL peptides (Example 5 below) and was exhibited evenafter the deletion of one N-terminal amino acid of the oligopeptide(Example 6 below).

Thus, when the oligopeptide is used for disease that collapses the motorneuron mechanism due to neuronal cell death caused by a SOD1 mutantgene, for example amyotrophic lateral sclerosis (ALS), the oligopeptidecan remedy the disease by inhibiting the neuronal cell death. As usedherein, the “motor neuron disease” refers to a neurodegenerative diseasewith progressive, retrograde disorder of upper and lower motor neuronsthat control motion in the body. Examples of the disease typicallyinclude amyotrophic lateral sclerosis (ALS) and also include, but notlimited to, spinal muscular atrophy (SMA: Werdnig-Hoffmann disease orKugelberg-Welander syndrome) and bulbospinal muscular atrophy (BSMA:Kennedy-Alter-Sung syndrome). The agent of the present invention hasefficacy as a preventive agent preventing or delaying the onset of themotor neuron disease and/or a therapeutic agent allowing the motorneuron disease to recover to the normal state. The agent of the presentinvention is also effective for the amelioration (or improvement) ofconditions resulting from the motor neuron disease. The amelioration ofconditions refers to the amelioration of, for example, muscular atrophy,muscular weakness, bulbar palsy (muscular atrophy or weakness in theface, pharynx, and tongue, and aphasia or dysphagia caused thereby),muscular fasciculation, and respiratory disorder.

The agent of the present invention can be provided as a pharmaceuticalcomposition by preparing a purified preparation of the oligopeptide intovarious types of dosage forms by a variety of methods known in the art.The agent of the present invention, when orally administered, may beprepared into tablets, capsule, granules, powders, pills, liquors forinternal use, suspensions, emulsions, syrups, or the like, or may bemade into dry products which are redissolved when used. Alternatively,the agent of the present invention, when parenterally administered, isprepared into intravenous injections (including infusion), intramuscularinjections, intraperitoneal injections, hypodermic injections,suppositories, or the like, and the agent used as a preparation forinjection is provided in the form of unit dose ampule or multiple dosecontainer.

Various types of these preparations can be produced by a routine methodby appropriately selecting excipients, fillers, binders, wetting agents,disintegrants, lubricants, surfactants, dispersants, buffers,preservatives, solubilizers, antiseptics, flavors, soothing agents,stabilizers, tonicity agents, and so on, typically used forpreparations. The content of the oligopeptide as an active ingredient inthe pharmaceutical composition may be on the order of, for example, 0.1to 10% by weight.

The agent of the present invention, when used as a preventive and/ortherapeutic agent for the disease described above, can be administeredparenterally or orally with safety to mammals such as humans, mice,rats, rabbits, dogs, and cats. The dose of the agent of the presentinvention may be changed appropriately depending on the ages ofindividuals to be administered, administration routes, and the number ofdoses. For example, the effective amount of the oligopeptide combinedwith suitable diluents and pharmacologically available carriers is, forexample, in the range of 1 to 500 μg/kg body weight/day.

The active ingredient of the agent of the present invention may be DNA(or a gene) encoding the oligopeptide. When the gene encoding theoligopeptide is used as a gene therapy agent for the disease describedabove, examples of administration methods thereof include a method whichdirectly administers the gene by injection and a method whichadministers a vector incorporating the gene therein. Examples of thevector include adenovirus vectors, adeno-associated virus vectors,herpes virus vectors, vaccinia virus vectors, and retrovirus vectors.Efficient administration can be achieved by using these virus vectors.Alternatively, a method which introduces the gene into phospholipidvesicles such as liposomes and administers the liposome may be used.

The administration mode of the gene-therapeutic agent may be any oflocal administration such as administration to quadriceps femoris muscleor gluteus maximus and systemic administration such as typicalintravenous or intraarterial administration and is preferably localadministration. Furthermore, the administration mode combined withcatheter techniques, surgical operation, and so on, can be adopted.

2. Expression System of DNA Encoding Oligopeptide

The oligopeptide used in the agent of the present invention can also beproduced according to typical genetic engineering techniques. Namely, arecombinant vector comprising the DNA encoding the oligopeptide isconstructed, and a microorganism (transformant) transformed with thevector is prepared. The desired oligopeptide can be separated andpurified from a culture obtained by culturing the transformant.

In the oligopeptide production by the genetic engineering techniques,the oligopeptide can be secreted actively outside of the host cell byexpressing it in the form of a fusion peptide of the oligopeptide with asignal peptide for extracellular secretion added to the N terminusthereof. Furthermore, a tag for purification/detection can be added tobetween the signal peptide and the oligopeptide or to the C-terminus ofthe oligopeptide.

The production of the fusion protein may be performed by procedures inwhich the DNA encoding the oligopeptide and DNA encoding another peptideare ligated in frame and introduced into an expression vector to expressthe fusion protein in a host. Approaches already known in the art can beused.

Any signal peptide of secretion proteins known in the art, which isselected depending on the types of host cells, can be used as the signalpeptide of the present invention. When animal cells are used as hostcells, examples of the signal peptide include signal peptides present inthe N termini of growth and differentiation factors (e.g., a variety ofcytokines) and receptors thereof.

Any of those known in the art can be used as the tag forpurification/detection, and examples thereof include FLAG, 6×His,10×His, influenza hemagglutinin (HA), VSV-GP fragments, T7-tag, HSV-tag,and E-tag.

Vectors containing DNAs encoding these peptide sequences as inserts andeach host (Escherichia coli, yeast, and animal cell) are commerciallyavailable.

The recombinant vector of the present invention can be obtained byligating the gene encoding the oligopeptide to an appropriate vector.The vector into which the gene is inserted is not particularly limitedas long as it enables replication in hosts. Examples of the vectorinclude plasmid DNA and phage DNA. Examples of the plasmid DNA includeEscherichia coli-derived plasmids, Bacillus subtilis-derived plasmids,and yeast-derived plasmids. Examples of the phage DNA include λ phages.Furthermore, animal viruses such as retrovirus or vaccinia virus andinsect virus vectors such as baculovirus can also be used.

To insert the gene into a vector, a method which initially cleaves thepurified DNA with appropriate restriction enzymes and inserts andligates it at the restriction site or multicloning site of appropriatevector DNA can be adopted.

The DNA encoding the oligopeptide is incorporated into the vector sothat the function of the DNA is exerted. Thus, the vector of the presentinvention can be ligated, if desired, with those containing cis elementssuch as enhancers, splicing signals, polyA-addition signals, selectivemarkers, ribosome-binding sequences (SD sequences), and so on, inaddition to promoters and the DNA. Examples of the selective markersinclude dihydrofolate reductase genes, ampicillin resistance genes, andneomycin resistance genes.

The host cell (i.e., transformant) of the present invention can beobtained by introducing the recombinant vector of the present inventioninto a host so that the DNA of interest can be expressed. In thiscontext, the host is not particularly limited as long as it can expressthe gene. Examples of the host include bacteria belonging to the genusEscherichia such as Escherichia coli and the genus Bacillus such asBacillus subtilis; yeasts such as Saccharomyces cerevisiae andSchizosaccharomyces pombe; and animal cells such as monkey COS-7 cell,Vero, Chinese hamster ovary cell (CHO cell), mouse L cell, human GH3cell, and human FL cell.

Examples of methods for introducing the recombinant vector into the hostinclude electroporation, calcium phosphate, lithium acetate,lipofection, and virus methods, which are selected depending on thetypes of hosts. Methods independent of recombinant vectors, for exampleparticle gun method, can also be used for gene delivery to each of thehost cells.

The oligopeptide can be obtained by culturing the transformant, followedby collection from the resulting culture. The “culture” means any ofculture supernatants, cultured cells or cultured microorganisms, andhomogenates of the cultured cells or cultured microorganisms.

The transformant of the present invention can be cultured in a mediumaccording to a method typically used for culturing the host cell.

Both natural and synthetic media may be used as the medium for culturingthe transformant obtained from a microorganism such as Escherichia colior yeast used as a host as long as the media contain carbon sources,nitrogen sources, inorganic salts, and so on capable of beingassimilated by the microorganism and can achieve the efficient cultureof the transformant. The carbon sources may be those capable of beingassimilated by the microorganism. Carbohydrates such as glucose,fructose, sucrose, and starch, organic acids such as acetic acid andpropionic acid, and alcohols such as ethanol and propanol can be used.Ammonium salts of inorganic or organic acids (e.g., ammonia, ammoniumchloride, ammonium sulfate, ammonium acetate, and ammonium phosphate),other nitrogen-containing compounds, peptone, meat extracts, corn steepliquor, and so on can be used as the nitrogen sources. Monopotassiumphosphate, dipotassium phosphate, magnesium phosphate, magnesiumsulfate, sodium chloride, ferrous sulfate, manganese sulfate, coppersulfate, calcium carbonate, and so on can be used as the inorganicsalts.

When the oligopeptide is produced into the cultured microorganism orcells, the oligopeptide is extracted by homogenizing the microorganismor cells. When the oligopeptide is produced outside of the microorganismor cells, the culture liquid is directly used or, for example,centrifuged to remove the microorganism or cells. Then, the oligopeptideof interest can be isolated and purified from the culture by using,alone or in appropriate combination, general biochemical methods used inprotein isolation and purification, for example ammonium sulfateprecipitation, gel chromatography, ion-exchange chromatography, andaffinity chromatography.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more fully withreference to Examples. However, the present invention is not intended tobe limited by these Examples.

All of experiments using neuronal cells lines described in Examplesbelow were repeated at least three times with independenttransformations and treatments, each of which gave essentially the sameresults. Statistical analysis was conducted by one-way ANOVA, followedby Bonferroni/Dunn post-hoc test, in which p<0.05 was assessed as beingsignificant.

Animal experiments were conducted according to Policies on the Use ofAnimals and Humans in Neuroscience Research, the Society forNeuroscience and Guideline for Care and Use of Laboratory Animals ofKEIO University. All experimental procedures were approved byInstitutional Animal Experiment Committee at KEIO University.

EXAMPLE 1 Inhibition of SOD1 Mutant Gene-Induced Neuronal Cell Death byADNF (1) Test Materials

Wild-type SOD1 cDNA (SEQ ID NO: 4) and SOD1 mutant (A4T-SOD1, G85R-SOD1,and G93R-SOD1) cDNAs were kindly provided by Dr. Shoji Tsuji (Faculty ofMedicine, the University of Tokyo). Kinase-inactive CaMKII and CaMKIVcDNAs were kindly provided by Dr. Howard Schulman, Stanford University,US. ADNF (SALLRSIPA: SEQ ID NO: 1) and ADNF8 (ALLRSIPA: SEQ ID NO: 2)were synthesized (Glazner G W, et al., 1999, J Neurochem 73: 2341-2347).IPAL peptide (IPALDSLKPANEDQKIGIEI: SEQ ID NO: 3, Zamostiano R, et al.,1999, Neurosci Lett 264: 9-12) was purchased from the Peptide Institute(Osaka, Japan). An anti-SOD1 antibody was purchased from MBL (Nagoya,Japan). PD98059, SB20380, AG490, KN93, KN92, and HA1004 were purchasedfrom Calbiochem-Novabiochem (San Diego, USA).

F11 cell, the hybrid cell of rat embryonic day 13 (E13) primary culturedneuronal cell with mouse neuroblastoma NTG18 cell, was cultured in Ham'sF-12 medium (Life Technologies, Gaithersburg, Md.) containing 18% FBS(Hyclone, Logan, Utah) and antibiotics as previously reported (PlatikaD, et al., 1985, Proc Natl Acad Sci USA 82: 3499-3503; Yamatsuji T, etal., 1996, Science 272: 1349-1352; Huang P, et al., 2000, Mol Hum Reprod6: 1069-1078; and Niikura T, et al., 2001, J Neuroscience, 21:1902-1910).

NSC34 cell, the hybrid cell of primary cultured, motor neuron-systemembryonic mouse spinal cord cell with mouse neuroblastoma NTG18 cell,was cultured in DEME medium containing 10% FBS and antibiotics (CashmanN R, et al., 1992, Dev Dyn 194: 209-221; and Durham H D, et al, 1993,Neurotoxicology 14: 387-395).

(2) Test Methods (2-1) Neuronal Cell Death Test

The F11 cells (7×10⁴ cells/well, 6-well plate, 12- to 16-hour culture inHam's F-12 (18% FBS) medium) were transformed with wild-type or SOD1mutant (A4T, G85R, and G93R) genes by lipofection (0.5 μg of each SOD1gene; 1 μl of LipofectAMINE; 2 μl of PLUS Reagent) under serum-freeconditions for 3 hours and cultured for 2 hours in Ham's F-12 (18% FBS)medium. The medium was replaced by Ham's F-12 (10% FBS) in the presenceor absence of ADNF. After 72 hours of the transformation, cell mortalitywas measured by Trypan blue exclusion assay as previously reported(Hashimoto Y, et al., 2001, J Neurosci 21: 9235-9245; and Hashimoto Y,et al, 2001, Proc Natl Acad Sci USA 98: 6336-6341).

The NSC34 cells (7×10⁴ cells/well, 6-well plate, 12 to 16-hour culturein DMEM (10% FBS) medium) were transformed with wild-type or SOD1 mutant(A4T, G85R, and G93R) genes by lipofection (0.5 μg of each SOD1 gene; 1μl of LipofectAMINE; 2 μl of PLUS Reagent) under serum-free conditionsfor 3 hours and cultured for 21 hours in DMEM (10% FBS) and after 48hours in DMED supplemented with N2 supplement (Invitrogen). After 72hours of the transformation, cell mortality was measured by Trypan blueexclusion assay in the same way as above.

(2-2) Immunoblot Analysis

Immunoblot analysis was conducted according to the previous report(Hashimoto Y, et al., supra, p. 6336-6341). To examine the proteinexpression of wild-type or SOD1 mutant genes, lysates from the cellstransfected with each SOD1 gene were subjected to SDS-PAGE (20 μg/lane).After electrical blotting to a PVDF membrane, the membrane was blockedby a typical method and reacted with an anti-SOD1 antibody and then witha 1:5000-diluted horseradish peroxidase-conjugated anti-mouse IgGantibody (Bio-Rad Lab. Hercules, Calif., USA). The antibody-reactivebands were detected by ECL (Amersham Pharmacia Biotech, Uppsala,Sweden).

(3) Result (3-1) Confirming Induction of Neuronal Cell Death of F11Cells by SOD1 Mutant Gene Transfer

F11 cells were transformed with 0.25, 0.5, or 1.0 μg of wild-type SOD1(wt SOD1) cDNA or SOD1 mutant (G85R-SOD1) cDNA. After 72 hours, cellmortality was measured by Trypan blue exclusion assay. The pEF-BOSvector was used as a control. Cell mortality induced by thetransformation with 0.25 or 0.5 μg of wild type SOD1 cDNA wasapproximately 10%, which was similar to that induced by thetransformation with the control vector. However, cell mortality inducedby the transformation with 1 μg of the wild type SOD1 cDNA was 45%,indicating that even wild-type SOD1 causes cell death by itsoverexpression (FIG. 1(A)). On the other hand, cell mortality induced bythe transformation with 0.25 μg of G85R-SOD1 cDNA was 17%, which wasslightly higher than that induced by the transformation with the controlvector, while cell mortalities induced by the transformation with 0.5and 1.0 μg of G85R-SOD1 cDNA were 50% and 60%, respectively (FIG. 1(B)).Based on these results, the amount of cDNA for the transformation ofthree types of SOD1 mutant genes to induce the cell death of F11 cellswas decided to be 0.5 μg.

(3-2) Effect of ADNF on Neuronal Cell Death Induced by SOD1 Mutant Gene

The effect of ADNF on neuronal cell death induced by SOD1 mutant geneswas tested. For comparison, S14G Humanin (HNG) was used. S14G-HN(HNG)has been reported to prevent neuronal cell death caused by someAlzheimer's disease-associated disorders but fail to prevent neuronalcell death induced by SOD1 mutant genes (Hashimoto Y, et al., supra, p.6336-6341).

F11 cells were transformed with wild-type or SOD1 mutant (A4T-SOD1,G85R-SOD1, and G93R-SOD1) genes (0.5 μg each) in the presence or absenceof 100 μM ADNF or 10 mM HNG. After 72 hours, cell mortality was measuredby Trypan blue exclusion assay. The pEF-BOS vector was used as acontrol.

The transformation with the SOD1 mutant genes resulted in the death of40 to 50% of the cells (FIG. 1(C)). On the other hand, thetransformation with the control vector caused the death of only 10% ofthe cells. The addition of 100 fM ADNF decreased the cell mortalitiesinduced by these SOD1 mutant genes to the level of the control. Bycontrast, the addition of 10 nM HNG could not reduce the cell deathinduced by the SOD1 mutant genes (FIG. 1(C)).

EXAMPLE 2 Dose-Dependent Inhibitory Effect of ADNF on Neuronal CellDeath

F11 or NSC34 cells were used to confirm the dose-dependent effect ofADNF on neuronal cell death induced by transformation with SOD1 mutantgenes (A4T-SOD1, G85R-SOD1, and G93R-SOD1).

F11 or NSC34 cells were transformed with pEF-BOS, A4T-SOD1, G85R-SOD1,or G93R-SOD1 cDNA in the presence of increasing concentrations (10 aM, 1fM, 100 fM, 10 pM, 1 nM, and 100 nM) of ADNF. After 72 hours, cellmortality was measured by Trypan blue exclusion assay.

Although 10 aM ADNF hardly exhibited cell death inhibition, 100 fM ADNFcompletely decreased cell mortalities induced by these three types ofSOD1 mutant genes to the level of the control (FIG. 2A). In this regard,the complete protective action of ADNF on cell death caused by the SOD1mutant genes was observed at the concentrations equal to or above 10 nM.These results demonstrated the dose-dependent inhibitory activity ofADNF against neuronal cell death caused by SOD1 mutant genes.

Similarly, the experiment using NSC34 cells also showed that 100 fM ADNFcan completely inhibit neuronal cell death caused by the three types ofSOD1 mutant genes (FIG. 2B). ADNF exhibited dose-dependent inhibitoryactivity against the neuronal cell death of NSC34 cells, as with the F11cells, and its effect was not decreased even at the concentrations equalto or above 10 nM.

Furthermore, the tests using both cells also demonstrated that SOD1 geneexpression levels are not affected by ADNF treatment.

ADNF has been reported to lose its neuroprotective action at or above 10nM (Brenneman D E, et al., 1996, J Clin Invest 97: 2299-2307; andBrenneman D E, et al., 1998, J Pharmacol Exp Ther 285: 619-627). Inagreement with this result, we have also confirmed that ADNF at or abovenM levels possesses reduced inhibitory activity against cell deathincluding neuronal cell death caused by amyloid β toxicity- or APPmutant gene-induced Alzheimer's disease-associated disorders (HashimotoY, et al., 2001, Proc Natl Acad Sci USA 98: 6336-6341).

Thus, the dose-dependent neuroprotective effect of ADNF confirmed in thepresent tests on neuronal cell death caused by SOD1 mutant was probablydifferent from that on amyloid β neurotoxicity.

EXAMPLE 3 Analysis on Neuroprotective Action of ADNF

Intracellular signaling by ADNF was examined. F11 cells were transformedwith pEF-BOS or SOD1 mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) cDNA andreacted with 10 nM wortmannin (PI3 kinase inhibitor), 100 μM genistein(tyrosine kinase inhibitor), 50 μM PD98059 (MEK inhibitor), 20 μMSB203580 (p38 MAPK inhibitor), 1 μM AG490 (JAK kinase inhibitor), 5 μMKN93 (calcium/calmodulin-dependent kinase inhibitor), or 10 μM HA1004(protein kinase A inhibitor) in the presence or absence of 100 nM ADNF.After 72 hours of the transformation, cell mortality was measured byTrypan blue exclusion assay.

The results obtained using A4T-SOD1, G85R-SOD1, and G93R-SOD1 arerespectively shown in FIGS. 3A to 3C.

The neuroprotective effect of ADNF was not affected by wortmannin (W),PD98059 (PD), SB203580 (SB), AG490 (AG), and HA1004 (HA), but it wasinhibited by genistein (G) and KN93 (KN) (FIGS. 3A to 3C). This suggeststhat ADNF exerts its neuroprotective action via certain tyrosine kinaseand calcium/calmodulin-dependent kinase (CaMK) but does not activate PI3kinase, MEK, p38 MAPK, JAK2, and PKA for exhibiting the neuroprotectiveaction.

EXAMPLE 4 Involvement of CaMKIV in Downstream of Signaling Pathway ofADNF

The effect of KN93 on the neuroprotective action of ADNF was examined.F11 cells were transformed with pEF-BOS or SOD1 mutant (A4T-SOD1,G85R-SOD1, or G93R-SOD1) cDNA and reacted with KN93 (10 nM, 50 nM, 100nM, 500 nM, 1 μM, or 5 μM) or 5 μM KN92, an inactive form of KN93, inthe presence or absence of 100 fM ADNF. After 72 hours of thetransformation, cell mortality was measured by Trypan blue exclusionassay.

The results obtained using A4T-SOD1, G85R-SOD1, and G93R-SOD1 arerespectively shown in FIGS. 4A to 4C.

The neuroprotective action of ADNF was partially inhibited by 50 nM KN93and almost completely inhibited by 1 μM KN93. KN93 at any of theconcentrations had no influence on cell mortality at the control level.As expected, inactive KN92 even at the concentration of 5 μM did notinfluence ADNF activity. The protein expression of SOD1 mutant detectedby immunoblot was not affected by KN93 or KN92. This dose-dependentinhibition of KN93 was also observed in all of the cells transformedwith A4T-SOD1, G85R-SOD1, and G85R-SOD1 (FIGS. 4A to 4C).

Next, the influence of a kinase-inactive form of CaMKII or CaMKIV on theneuroprotective action of ADNF was examined.

NSC34 cells were cotransformed with pSR-alpha, A4T-SOD1, G85R-SOD1 orG93R-SOD1 cDNA and with pSR-alpha, a kinase-inactive CaMKII cDNA, or akinase-inactive CaMKIV cDNA, and further cultured in the presence orabsence of 100 nM ADNF. After 72 hours from the transformation, the cellmortality was measured.

Neither of the control vector or the kinase-inactive CaMKII influencedthe neuroprotective action of ADNF. By contrast, the kinase-inactiveCaMKIV inhibited the neuroprotective action of ADNF (FIG. 4D). Thissuggests that CAMKIV, but not CaMKII, is located downstream of thesignaling pathway of ADNF. Neither the kinase-inactive CaMKII nor thekinase-inactive CaMKIV influenced the cell mortality at the controllevel.

EXAMPLE 5 Difference Between Neuroprotective Effect of ADNF onFALS-Associated Disorders and Protective Effect on Amyloid β Toxicity

The IPAL peptide (IPALDSLKPANEDQKIGIEI: SEQ ID NO: 3) has been reportedto inhibit the protective effect of ADNF on the cell death induced byamyloid β (Zamostiano R, et al., 1999, Neurosci Lett 264: 9-12). Thus,the effect of the IPAL peptide on the neuroprotection of ADNF againstcell death caused by SOD1 mutant genes was examined.

F11 cells were transformed with pEF-BOS or SOD1 mutant (A4T-SOD1,G85R-SOD1, or G93R-SOD1) cDNA and reacted with 100 μM ADNF in thepresence of 10 μM IPAL peptide. After 72 hours of the transformation,cell mortality was measured. The IPAL peptide did not inhibit theinhibitory activity of ADNF against cell death caused by the three typesof SOD1 mutant genes, and it did not influence the cell death caused bythe SOD1 mutant genes (FIG. 5A). In this regard, the IPAL peptide itselfdid not influence the cell death at the control level.

EXAMPLE 6 Effect of N-Terminally Truncated ADNF

ADNF8 (ALLRSIPA: SEQ ID NO: 2), which lost one N-terminal amino acidresidue in ADNF, has been reported to be totally ineffective againstneuronal cell death caused by amyloid β or TTX (Brenneman D E, et al.,1998, J Pharmacol Exp Ther 285: 619-627). Thus, the effect of ADNF8 oncell death caused by G93A-SOD1 was examined.

F11 cells were transformed with pEF-BOS or G93A-SOD1 cDNA and reactedwith varying concentrations of ADNF (SALLRSIPA) or ADNF8 (ALLRSIPA).After 72 hours of the transformation, cell mortality was measured. ADNF8at the concentration of 10 μM completely inhibited cell death caused byG93R-SOD1, indicating that ADNF8 is effective, though 100-fold less thanADNF, for the inhibition of neuronal cell death caused by SOD1 mutantgenes (FIG. 5B).

EXAMPLE 7 Motor Performance Test with ALS Model Animal

(1) Animal used

Transgenic (Tg) mice (hereinafter, referred to as “G93A-SOD1 Tg mice”)expressing human FALS-associated SOD1 mutant gene with a mutation (G93A)from Gly to Ala at 93 position are the best-established mouse model ofALS (Gurney M E, et al., 1994, Science 264: 1772-1775; and Gurney M E,et al., 1997, J Neurol Sci 152 Suppl 1: S67-73). The G93A-SOD1 Tg micemanifest symptoms quite similar to human ALS after normal birth andrapidly result in death in all cases. The G93A-SOD1 Tg mice, which havethe onset of the regression of motor neurons similar clinically andpathologically to that in human ALS, have been known so far to be themost excellent model of ALS throughout the world and employed foridentifying effective candidate agents for ALS patients.

(2) Test methods

The model mice were used to confirm the in vivo effect of ADNF onneurotoxicity induced by G93A-SOD1.

The G93A-SOD1 Tg mice were purchased from Jackson Laboratories (BarHarbor, Me.). The G93A-SOD1 Tg mice were kept as hemizygote mice by themating thereof with C57BL/6J mice (CLEA Japan, Inc). The mice wereraised in a SPF room (specific pathogen-free animal facility; 23±1° C.,55±5% humidity) in the 12-hour light/12-hour dark cycle (7:00 AM-7:00PM). The mice were freely fed with gamma ray-irradiated Picolab RodentDiet 20 (PMI Feeds Inc. St. Louis, Mo.) and sodium hyposulfite (5ppm)-containing aseptic deionized distilled water.

The G93A-SOD1 Tg mice at 10 weeks of age were put under anesthesia bythe intraperitoneal injection of 10% sodium pentobarbital (60 mg/kg). Ahole was made on the cranial bone with a drill on the operating table ofa stereotactic instrument to aseptically transplant the C315GS-4 cannulasystem for mouse (Plastics One Inc., Roanoke, Va.) to the mouse leftcerebral ventricle. The cannula was fixed with a surgical adhesive anddental cement. The mice at 80 days of age were divided at random to asaline (control)-administered group (n=8) and an ADNF-administered group(n=1), and 3 μl of saline and 3 μl of 30 nmol ADNF wereintracerebroventricularly (icv) injected each day to the former and thelatter groups, respectively, until the end of the experiment. Theinjection was performed with the cannula in C3151S-4 connected toHamilton syringe through a cannula tube (C232, PE50/Thin wall, PlasticOne).

The motor function (motor performance) was evaluated weekly with arotarod (CLEA Japan Inc). After the cannulation, the mice wereacclimated to the rotarod for 2 days. The mice were placed onto arotating rod moving at 5 rpm, and the time for which each mouse couldremain on the rod was automatically detected. The test was conductedaccording to the protocol wherein the test was completed as a score of 7minutes if the mouse remained on the rod for 7 minutes (Li M, et al.,2000, Science 288: 335-339; and Kaspar B K, et al., 2003, Science 301:839-842). Disease onset was defined as the first day when the mousecould not remain on the rotarod for 7 minutes.

Death was defined when the mouse was unable to right itself within 30seconds after being placed on its back (Li M, et al., supra).

(3) Result

The ADNF-treated ALS mice had motor performance significantly betterthan that of the control mice on Week 16, suggesting that theADNF-treated mice did not cause reduction in motor function andmaintained their motor functions (FIG. 6). However, this differencebetween the control mice and the ADNF-treated mice disappeared on Week18 to Week 20.

The disease onset and survival days of the saline- and ADNF-administeredgroups are shown in Table 1 below.

TABLE 1 Saline-administered ADNF-administered group (n = 8) group (n =11) Disease onset days 122.5 ± 7.2 127.3 ± 2.6 Survival days 156.8 ± 3.5155.3 ± 2.2

Disease onset in the ADNF-administered group was prone to be delayed,although no significant difference in disease onset was obtained betweenthe saline-administered group and the ADNF-administered group. The meansurvival days of the ALS control mice and the ADNF-treated group were156.8±3.5 days and 155.3±3.7 days, respectively, showing no differencein survival rate between them.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention provides an agent that significantly inhibits (orantagonizes) neuronal cell death induced by superoxide dismutase-1(SOD1) mutant genes and has the effect of delaying the onset of motorneuron diseases including amyotrophic lateral sclerosis (ALS) andimproving the motor function of a patient with motor neuron disease.Thus, the agent of the present invention is useful for the treatmentand/or prevention of motor neuron diseases.

1. A therapeutic and/or preventive agent for motor neuron diseasecomprising an oligopeptide shown in any of the following (a) to (c) or apharmaceutically acceptable salt thereof as an active ingredient: (a) anoligopeptide consisting of the amino acid sequence represented bySer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); (b) an oligopeptideconsisting of an amino acid sequence having a deletion, substitution,insertion, or addition of one or several amino acids inSer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and having anactivity that inhibits neuronal cell death caused by a mutant superoxidedismutase-1 gene; and (c) a modified oligopeptide from the oligopeptide(a) or (b).
 2. A therapeutic and/or preventive agent for motor neurondisease comprising DNA encoding the following oligopeptide (a) or (b) asan active ingredient: (a) an oligopeptide consisting of the amino acidsequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1); and (b) an oligopeptide consisting of an amino acid sequence havinga deletion, substitution, insertion, or addition of one or several aminoacids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and havingan activity that inhibits neuronal cell death caused by a mutantsuperoxide dismutase-1 gene.
 3. The agent according to claim 1 or 2,wherein the motor neuron disease is amyotrophic lateral sclerosis (ALS).4. The agent according to claim 1 or 2, wherein the agent has the effectof delaying the onset of the motor neuron disease and ameliorating themotor function of a patient with the motor neuron disease.
 5. A fusionpeptide of the following oligopeptide (a) or (b) with another peptide:(a) an oligopeptide consisting of the amino acid sequence represented bySer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); or (b) anoligopeptide consisting of an amino acid sequence having a deletion,substitution, insertion, or addition of one or several amino acids inSer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and having anactivity that inhibits neuronal cell death caused by a mutant superoxidedismutase-1 gene.
 6. The fusion peptide according to claim 5, whereinanother peptide is a signal peptide for extracellular secretion and/or atag peptide for purification and detection.
 7. DNA encoding thefollowing oligopeptide (a) or (b) or a fusion peptide according to claim5: (a) an oligopeptide consisting of the amino acid sequence representedby Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); or (b) anoligopeptide consisting of an amino acid sequence having a deletion,substitution, insertion, or addition of one or several amino acids inSer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and having anactivity that inhibits neuronal cell death caused by a mutant superoxidedismutase-1 gene.
 8. A recombinant vector comprising DNA according toclaim
 7. 9. A host cell transformed with DNA according to claim
 8. 10. Amethod for producing the following oligopeptide (a) or (b),characterized by culturing the host cell according to claim 9 in amedium and collecting an oligopeptide expressed from the obtainedculture: (a) an oligopeptide consisting of the amino acid sequencerepresented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1): and(b) an oligopeptide consisting of an amino acid sequence having adeletion, substitution, insertion, or addition of one or several aminoacids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and havingan activity that inhibits neuronal cell death caused by a mutantsuperoxide dismutase-1 gene.